Bacterial resistance to antibiotics is a serious medical problem. Treatment including bacteriophages might have an effective alternative that is known for a long time but has been ignored outside the former Soviet Union. Appropriately selected phages can easily be used to help prevent bacterial diseases in human’s o animals, with potential for alternative applications and special interest for developing countries.

BACKGROUND

In all corners of the world, the resistance of bacteria to antibiotics is becoming a very serious medical problem. Whenever antibiotics are used, the development of resistance is a logical consequence; as all other living organisms, the bacteria adapts to the environmental conditions in a continuous process of evolution. The question is not whether antibiotic resistance will occur, but when.
Ironically, the resistance builds up by both insufficiency of dose as well as the overuse of antibiotics. In industrialized countries, bacteria its developing resistance to a variety of antibiotics, a process that threatens to make the achievements of modern medicine useless. Without the protection against bacterial infections, for example, extensive operations and treatments that weaken the patients’ immune system, such as organ transplantation or chemotherapy, would not be feasible.
In the past 30 years, new classes of antibiotics were not found. Even the modern biotechnology could not salvage with its genetic engineering. Pharmaceutical companies have mainly focused on the development of new products relying only known classes of antibiotics.

THE NEED FOR AN ALTERNATIVE TO ANTIBIOTICS

In the United States, at least 2 million people annually become infected with bacteria that is resistant to antibiotics. At least 23,000 people die each year as a direct result of the infections, while many others die from other conditions that are the complications of an antibiotic-resistant infection (Frieden 2013)
The appearance of infectious diseases caused by drug-resistant bacteria requires alternatives to conventional antibiotics (Barrow and Soothill 1997; Alisky et al. 1998; Carlton 1999; Sulakvelidze et al. 2001). The quest for new drugs is becoming more and more critical because of the growing concern over the failing antibiotic medicine discovery pipeline. There is a lot of interest to find alternatives and natural antimicrobial agents, which has also grown due to consumer awareness about the use of chemical preservatives in food.

Bacteriophages, also known as phages, are viruses that infect bacteria. These phages also require a bacterial host in order to multiply themselves. These bacterial viruses are made up of proteins that coat an inner core of nucleic acid – either DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). Phages also vary in their structure; ranging from the simple to the more complicated.

The way the bacteriophage works:
So as to infect a host cell, the bacteriophage attaches itself to the bacteria’s cell wall, specifically on a receptor located on the bacteria’s surface. After it connects firmly to the cell, the bacterial virus injects its genetic material (its nucleic acid) into the hosting cell. Depending on the type of phage, one of two cycles will occur – The Lytic or The Lysogenic cycle. During a lytic cycle, the phage will make use of the host cell’s chemical energy as well as its biosynthetic machinery in order to produce phage nucleic acids (phage DNA and phage mRNA) and phage proteins. After the production chapter is finished, the phage nucleic acids and structural proteins are assembled. Eventually, certain proteins made inside the cell will cause the cell wall to lyse, allowing the assembled phages within to be release and to infect other bacterial cells.

Viral reproduction can also appear through the lysogenic cycle. The main difference between the two types of cycles is that during lysogeny, the host cell is not destroyed or does not undergo lysis. Once the host cell is infected, the phage DNA blends or fuses with the bacterial chromosome, constructing the prophage. When the bacterium reproduces, the prophage is replicated along with the host chromosomes. Thus, the daughter cells also contain the prophage which carries the ability to producing phages. The lysogenic cycle can continue endlessly (daughter cells with prophage present within continuing to replicate) unless exposed to adverse conditions which can start the termination of the lysogenic phase and cause the expression of the phage DNA and the start of the lytic cycle. These adverse conditions include exposure to UV or mutagenic chemicals and desiccation.

Phage therapy or viral phage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections. Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture. If the target host of a phage therapy treatment is not an animal the term "biocontrol" (as in phage-mediated biocontrol of bacteria) is usually employed, rather than "phage therapy".
Phages are currently being used therapeutically to treat bacterial infections that do not respond to conventional antibiotics, particularly in Russia and Georgia. There is also a phage therapy unit in Wroclaw, Poland, established 2005, the only such center in European Union countries.

Also phage treatment in human eyes, ears and nose via inhalation was used at the Eliava Institute in Tbilisi for decades (Kutter et al. 2010; Abedon et al. 2011). Recently, phages have been suggested to be included in a nebulizer to treat bacterial lung infections in cystic fibrosis patients (Golshahi et al. 2008) or to be sprayed as dried phages in respirable powders for the treatment of pulmonary infections (Matinkhoo et al. 2011).

Bacteriophage treatment offers a possible alternative to conventional antibiotic treatments for bacterial infection. It is conceivable that, although bacteria can develop resistance to phage, the resistance might be easier to overcome than resistance to antibiotics. Just as bacteria can evolve resistance, viruses can evolve to overcome resistance; however, the ability to evolve raises serious safety questions.
Bacteriophages are very specific, targeting only one or a few strains of bacteria. Traditional antibiotics have more wide-ranging effect, killing both harmful bacteria and useful bacteria such as those facilitating food digestion. The specificity of bacteriophages might reduce the chance that useful bacteria are killed when fighting an infection.

Bacteriophages are natural antibacterials able to regulate bacterial populations by the induction of bacterial lysis. They are active against gram-positive, as well as gram-negative bacteria, including MDR pathogens. actually, as mechanism of action phage lysis is totally different from antibiotics, retaining activity against bacteria exhibiting multiple mechanisms of antibiotic resistance. Because of its specificity, phage therapy has a narrow antibacterial spectrum with an effect limited to one single species or in some cases a single strain within a species. This limits the “pressure” and the heavy collateral damage done to bystander, non-targeted bacteria from antibiotics. The entire microbiome of the patient is altered by antibiotics, not just the intended target pathogen. In contrast, Chibani-Chennoufi et al. demonstrated little impact on the gut microbiota in mice after oral administration of phage therapy directed against E. coli. Preservation of much of the existing microbiome during phage therapy has been confirmed in careful microbial surveys in adult healthy volunteers who ingested a 9-phage cocktail. Phage therapy also avoids the potential overgrowth of secondary pathogens.
Since large, randomized, controlled trials are lacking at the present time, it is difficult to evaluate side effects and their potential impact. Based on the reports gained from Poland and the former Soviet Union, phage therapy seems to be without significant adverse effects; the fact that bacteriophages interact with bacterial cells only and do not interfere with mammalian cells probably could potentially explain this lack of deleterious side effects. Underreporting could be another explanation. However, the excellent tolerability of phage treatment has been demonstrated in preclinical studies in various animal models and in several observational studies in patients and healthy human volunteers. There is a wide distribution of phages upon systemic administration, including the ability to penetrate the blood brain barrier, allowing these agents to be used in case of central nervous system infections. Interestingly, at least some phages also display the capacity to disrupt bacterial biofilms.

Phage therapy may have an impact on the inflammatory response to infection. In 51 patients presenting with various long-term suppurative infection, TNFα release, in vivo and in vitro upon stimulation with LPS, was attenuated based upon the initial pattern of serum TNFα level. Release of IL-6 was only significantly reduced in vivo. C-reactive protein and white blood cell count were initially not affected in this patient population while it significantly decreased between day 9 and day 32 in 37 patients given oral phage therapy for osteomyelitis, prosthetic joint infection, skin and soft tissue infections, and, in one case, lung infection. This was an observational study without a control group and therefore should be cautiously interpreted. In a more recent observation, CRP was only affected in patients whose initial CRP serum level was above 10 mg/dl. White blood cells may also be affected by phage therapy: increased neutrophil precursors and decreased phagocytic index for Staphylococcus aureus was observed in patients after 3 weeks and 3 months of therapy, as compared with healthy donors. A large review of the alteration of immune responses with phage therapy has recently been published.wikipedia

HUMAN APPLICATIONS

The first report on the use of bacteriophage in humans described its efficacy in staphylococcal skin furuncles16 and d’Herelle summarized all his clinical work in 1931.
However, as already described, the enthusiasm for phage therapy declined in the western countries in the 1930s because of the questions regarding scientific rigor in testing phage therapy in the reports by Eaton and colleagues7-9 and also as a consequence of the discovery and the ease of use of antibiotics. The use of bacteriophages continued in the eastern countries and large number of reports were published over time, mainly in Poland and Georgia (former USSR).
Indeed, the first phase I randomized controlled trial conducted in the United States was published in 2009.31 It evaluated the safety of a cocktail of phages directed against E. coli, S. aureus, and Pseudomonas aeruginosa in 42 patients with chronic venous leg ulcers. The study was not powered to detect any positive outcome such as rate or frequency of healing but the authors did not find any adverse event related to the treatment. Another randomized trial was conducted in the UK and studied the efficacy of one application of a solution containing 6 bacteriophages in the ears of patients suffering chronic Pseudomonas aeruginosa-related otitis. The colony counts of P. aeruginosa significantly decreased in the treated group in this well done, double-blind, placebo-controlled study while various subjective clinical indicators improved in those patients. Indeed, patients reported lower intensity of symptoms such as discomfort, itching, wetness, and unpleasant odor. Likewise, physicians in charge of the patients (and blinded to the assigned treatment) reported decreased clinical observations such as erythema/inflammation, ulceration/granulation/polyps, and odor. There were no reported adverse reactions.

A small phase I study of 9 patients treated at the Burn Wound Centre of the Queen Astrid Military Hospital, Brussels, Belgium, was recently performed. Patients were locally treated with the BFC-1 phage cocktail containing 3 lytic phages: a Myovirus, a Podovirus against Pseudomonas aeruginosa, and a Myovirus directed against Staphylococcus aureus. A large burned section was exposed to a single spray application while a distant portion of the wound served as control. While complete results are yet to be published, there was no safety issue reported.
Finally, a randomized controlled trial confirmed the safety of an orally administered phage solution in healthy non-infected patients.

The advantages of phage therapy over antibiotic therapy: 1. it is effective against multidrug-resistant pathogenic bacteria; 2. substitution of the normal microbial flora does not occur because the phages kills only the targeted pathogenic bacteria;3. it can respond quickly to the appearance of phage-resistant bacterial mutants because the frequency of phage mutation is significantly higher than that of bacteria; 4. developing costs for a phage treatment is cheaper than that of new antibiotics; and 5. side-effects are very rare.

However, there are still some concerns such as:
1. rapid cell lysis of bacteria may result in the release of large amount of bacterial membrane-bound endotoxins; 2. some phages may encode toxins; 3. lack of pharmacokinetic data; 4. neutralization of phages by the host immune system may lead to failure of phage therapy; 5. conversion of lytic phages to lysogenic phages (prophages) leads to bacterial immunity to attacks by the corresponding lytic phages and may also change the virulence of the bacteria.

Despite the large number of publications on phage therapy, there are very few reports in which the pharmacokinetics of therapeutic phage preparations is delineated (Payne et al. 2000; Robert et al. 2000; Payne and Jansen 2003; Levin and Bull 2004; Brüssow 2005; Górski et al. 2006; Gill 2008; Cairns et al. 2009; Abedon and Thomas-Abedon 2010; Gill 2010; Abedon et al. 2011; Parracho et al. 2012). The studies of Bogovazova et al. (1991) and Bogovazova et al. (1992) suggested that phages get into the bloodstream of laboratory animals (after a single oral dose) within 2–4 h and that they are found in the internal organs (liver, spleen, kidney, etc.) in approximately 10 h. Also, data concerning the persistence of administered phages indicate that phages can remain in the body for relatively prolonged periods of time, i.e., up to several days (Babalova et al. 1968). In one study, the time needed for the phage to reduce, eliminate or cure the target bacteria in infected animals was defined as a reduction of Salmonella concentration in the chicken cecum, and obtained when the phage was administered one day before or just after bacterial infection and then again on different days post-infection (Bardina et al. 2012). In comparison, calves and piglets with diarrhea due to experimentally administered pathogenic E. coli were cured within 8 h following phage administration (Smith and Huggins 1983). Hence, elimination of the pathogenic E. coli at the pre-harvest stage could play a significant role in preventing its introduction into the food chain (Tauxe 1997). These results would suggest that due to the phage short-term effect; the application would be optimized according to the type of chronic infection with the length of time before slaughter that is required to control the particular infection for the animals.

D'Herelle's commercial laboratory in Paris produced at least five phage preparations against various bacterial infections. The preparations were called Bacté-coli-phage, Bacté-rhino-phage, Bacté-intesti-phage, Bacté-pyo-phage, and Bacté-staphy-phage, and they were marketed by what later became the large French company L'Oréal . Therapeutic phages were also produced in the United States. In the 1940s, the Eli Lilly Company (Indianapolis, Ind.) produced seven phage products for human use, including preparations targeted against staphylococci, streptococci, Escherichia coli, and other bacterial pathogens. These preparations consisted of phage-lysed, bacteriologically sterile broth cultures of the targeted bacteria (e.g., Colo-lysate, Ento-lysate, Neiso-lysate, and Staphylo-lysate) or the same preparations in a water-soluble jelly base (e.g., Colo-jel, Ento-jel, and Staphylo-jel). They were used to treat various infections, including abscesses, suppurating wounds, vaginitis, acute and chronic infections of the upper respiratory tract, and mastoid infections. However, the efficacy of phage preparations was controversial , and with the advent of antibiotics, commercial production of therapeutic phages ceased in most of the Western world. Nevertheless, phages continued to be used therapeutically—together with or instead of antibiotics—in Eastern Europe and in the former Soviet Union. Several institutions in these countries were actively involved in therapeutic phage research and production.

Phage therapy has many benefits in the humane medicine . the fact that bacteriophages could have an improved efficacy as compared with antibiotics provides the greatest hope for the future.
As yet phage clinical therapy is in a progressive and scientific cumulative stage. To move into the pharmaceutical stage, it needs competencies, best practices and data that can support pharmaceutical industries to develop phage therapy ‘live drugs’.
However, further well-conducted studies are required to define the role and safety of phage therapy in daily clinical practice to treat patients with various infections.